Selection of RNA aptamers against human influenza virus hemagglutinin using surface plasmon resonance

Aptamers are functional nucleic acids possessing high affinity and specificity to their cognate ligands and are isolated from a library of nucleic acids by iterative rounds of selection and amplification. In the current study, we used surface plasmon resonance (Biacore) as an efficient methodology for selecting aptamers that bind to hemagglutinin (HA) of human influenza virus. This procedure allowed us to monitor and select the target-bound aptamers specifically and simultaneously. These studies not only yielded an aptamer that binds to the HA of influenza virus with high affinity but also revealed the consensus sequence, 5'-GUCGNCNU(N)(2-3)GUA-3, for HA recognition.     

Oligomerization paths of the nucleoprotein of influenza A virus

The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.     

Assays for cytokines using aptamers

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.     

Determinants of apical loop-internal loop RNA-RNA interactions involving the HCV IRES

Domain II of the hepatitis C virus internal ribosome entry site is a major RNA structure involved in the viral mRNA translation. It comprises four different structural domains. We performed in vitro selection against the apical loop of the domain II and we identified RNA aptamers folding as an imperfect hairpin with an internal loop of interacting with the apical loop of the domain II. This RNA-RNA interaction creates apical loop-internal loop complex. The aptamer binds the target with an apparent K(d) of 35nM. In this study, the main structural elements of the target and the aptamer involved in the formation of the complex are characterized by mutation, deletion, and RNase probing analysis. We demonstrate that a complementary loop flanked by G,C rich upper and lower stems are crucial for such RNA-RNA interactions.     

RNA aptamers directed against release factor 1 from Thermus thermophilus

An in vitro selection/amplification (SELEX) was used to generate RNA aptamers that specifically bind Thermus thermophilus release factor 1 (RF1). From 31 isolated clones, two groups of aptamers with invariable sequences 5'-ACCU-3' and 5'-GAAAGC-3' were isolated. Chemical and enzymatic probing of the structure indicate that in both groups the invariable sequences are located in single-stranded regions of hairpin structures. Complex formations between RF1 and aptamers of both groups were identified by electrophoretic shift assay and chemical footprinting. Deletion of the invariable sequences did not effect the secondary structure of the aptamers but abolished their binding to RF1. RNA motifs matching the invariable sequences of the aptamers are present as consensus sequences in the peptidyl transferase center of 23S rRNAs. T. thermophilus RF1 recognizes UAG stop codons in an Escherichia coli in vitro translation system. Aptamers from both groups inhibited this RF1 activity.     

Direct selection of RNA beacon aptamers

A method for the direct selection of RNA molecules that can be easily converted into beacon aptamers is presented. Beacon aptamers are fluorescently labeled nucleic acids that signal the presence of a specific ligand through changes in fluorescence intensity. Typically, ligand binding causes an increase in fluorescence intensity by inducing a conformational change that separates a fluorophore/quencher pair. The method presented here simultaneously selects for ligand binding and induction of an appropriate conformational change. The method was tested by selecting RNA molecules that can detect the aminoglycoside antibiotic tobramycin. After 14 rounds of selection, two sequence families emerged. Upon conversion into beacon aptamers, representatives of the two selected sequence families specifically detected tobramycin, while a negative control RNA that did not survive the selection protocol did not function as a tobramycin beacon aptamer.     

Bioinformatic Analysis of the Contribution of Primer Sequences to Aptamer Structures

Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust.     

Assignment methodology for larger RNA oligonucleotides: Application to an ATP-binding RNA aptamer

The use of uniform 13C,15N labeling in the NMR spectroscopic study of RNA structures hasgreatly facilitated the assignment process in small RNA oligonucleotides. For ribose spinsystem assignments, exploitation of these labels has followed previously developed methodsfor the study of proteins. However, for sequential assignment of the exchangeable andnonexchangeable protons of the nucleotides, it has been necessary to develop a variety of newNMR experiments. Even these are of limited utility in the unambiguous assignment of largerRNAs due to the short carbon relaxation times and extensive spectral overlap for all nuclei.These problems can largely be overcome by the additional use of base-type selectively13C,15N-labeled RNA in combination with a judicious use of related RNAs with basesubstitutions. We report the application of this approach to a 36-nucleotide ATP-binding RNAaptamer in complex with AMP. Complete sequential 1H assignments, as well as the majorityof 13C and 15N assignments, were obtained.     

Epigenetic effects in eukaryotic gene expression

In the broadest terms, epigenetic phenomena in eukaryotes depend on the interaction of alleles or repeated sequences or on the mitotic inheritance of chromatin states or methylation patterns. One of the most exciting aspects of the study of epigenetic phenomena is the insight that can be gained into the structure and assembly of higher-order chromatin structures, an important subject that has proved refractory to current biochemical methodologies. Rapid progress in the study of gene inactivation in fungi, plants, and invertebrates will provide new hypotheses to be tested in mammals. © 1994 Wiley-Liss, Inc.     

Computational approaches toward the design of pools for the in vitro selection of complex aptamers

It is well known that using random RNA/DNA sequences for SELEX experiments will generally yield low-complexity structures. Early experimental results suggest that having a structurally diverse library, which, for instance, includes high-order junctions, may prove useful in finding new functional motifs. Here, we develop two computational methods to generate sequences that exhibit higher structural complexity and can be used to increase the overall structural diversity of initial pools for in vitro selection experiments. Random Filtering selectively increases the number of five-way junctions in RNA/DNA pools, and Genetic Filtering designs RNA/DNA pools to a specified structure distribution, whether uniform or otherwise. We show that using our computationally designed DNA pool greatly improves access to highly complex sequence structures for SELEX experiments (without losing our ability to select for common one-way and two-way junction sequences).     

Kinetic analysis and fragment screening with Fujifilm AP-3000

We evaluated the performance of Fujifilm’s new AP-3000 surface plasmon resonance biosensor for kinetic analysis and fragment screening. Using carbonic anhydrase II as a model system, we characterized a set of 10 sulfonamide-based inhibitors that range in molecular mass from 98 to 341 Da and approximately 10,000-fold in affinity (0.4 mM to 20 nM). Although the data collected from the AP-3000 were generally similar to those collected using a Biacore T100, the AP-3000’s stop-flow analyte delivery system complicated the shapes of the association- and dissociation-phase binding responses. We illustrate how reasonable estimates of the kinetic rate constants can be extracted from AP-3000 data by limiting data analysis to only the regions of the responses collected during flow conditions. We also provide an example of the results obtained for a fragment-screening study with the AP-3000, which is the ideal application of this technology.     

The Robustness of Naturally and Artificially Selected Nucleic Acid Secondary Structures

Thermodynamic stability and mutational robustness of secondary structure are critical to the function and evolutionary longevity of RNA molecules. We hypothesize that natural and artificial selection for functional molecules favors the formation of structures that are stable to both thermal and mutational perturbation. There is little direct evidence, however, that functional RNA molecules have been selected for their stability. Here we use thermodynamic secondary structure prediction algorithms to compare the thermal and mutational robustness of over 1000 naturally and artificially evolved molecules. Although we find evidence for the evolution of both types of stability in both sets of molecules, the naturally evolved functional RNA molecules were significantly more stable than those selected in vitro, and artificially evolved catalysts (ribozymes) were more stable than artificially evolved binding species (aptamers). The thermostability of RNA molecules bred in the laboratory is probably not constrained by a lack of suitable variation in the sequence pool but, rather, by intrinsic biases in the selection process.     

An RNA aptamer that recognizes a specific conformation of the protein calsenilin

The generation of molecules that selectively recognize specific conformations of a protein is an important component of the elucidation protein function. We have used SELEX (Systematic Evolution of Ligands by EXponential enrichment) technology to produce aptamers that bind in a conformationally selective manner to calsenilin, which involved in Ca²⁺-mediated apoptotic signaling. Since the conformations of calsenilin are quite different in the presence and absence of Ca²⁺, aptamers were selected against the dimeric protein both under calcium-bound and calcium-free conditions. We have found that aptamer-12 selectively binds to the dimeric form of the protein in the presence of calcium ion, while the binding of aptamer-2 does not discriminate between the Ca²⁺ bound and unbound protein. Data obtained from biochemical and biophysical experiments suggest that a dominant conformation of calcium-bound calsenilin exists in one dominant conformation and that one aptamer can be generated to recognize this conformation. In addition, observation made in this effort that aptamers selected against the two different conformations of calsenilin have different characteristics suggest that aptamers can serve as a plausible tool for recognizing various conformations of proteins, even those caused by interactions with small molecules or ions such as Ca²⁺.     

Application of Microchip Electrophoresis in the Analysis of RNA Aptamer-Protein Interactions

DNA and RNA can be separated by microchip electrophoresis (ME) and detected using an intercalating fluorescent dye. The advantages of this method are short sensing times (< 3 min), avoidance of a radioisotope labeling detection system, relatively low costs, and reduced labor intensity. In the present study, RNA aptamer-protein or -peptide interactions were analyzed using ME and the regression of free aptamers corresponding to unbound RNA was detected as the target protein or peptide increased in a dose-dependent manner. Our results demonstrate the applicability of this method to simple, rapid ligand screening in the interactions between oligonucleotides and their targets.     

Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 × 10⁻⁶ M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl--d-mannopyranoside.     

Conditional gene expression by controlling translation with tetracycline-binding aptamers

We present a conditional gene expression system in Saccharomyces cerevisiae which exploits direct RNA–metabolite interactions as a mechanism of genetic control. We inserted preselected tetracycline (tc) binding aptamers into the 5′-UTR of a GFP encoding mRNA. While aptamer insertion generally reduces GFP expression, one group of aptamers displayed an additional, up to 6-fold, decrease in fluorescence upon tc addition. Regulation is observed for aptamers inserted cap-proximal or near the start codon, but is more pronounced from the latter position. Increasing the thermodynamic stability of the aptamer augments regulation but reduces expression of GFP. Decreasing the stability leads to the opposite effect. We defined nucleotides which influence the regulatory properties of the aptamer. Exchanging a nucleotide probably involved in tc binding only influences regulation, while mutations at another position alter expression in the absence of tc, without affecting regulation. Thus, we have developed and characterized a regulatory system which is easy to establish and controlled by a non-toxic, small ligand with good cell permeability.     

Regulating gene expression with light-activated oligonucleotides

Since the development of light-responsive amino acids, the activity of numerous biomolecules has been photomodulated in biochemical, biophysical, and cellular assays. Biological problems of even greater complexity motivate the development of quantitative methods for controlling gene activity with high spatial and temporal resolution, using light as an external trigger. Photoresponsive DNA and RNA oligonucleotides would optimally serve this purpose, but have proven difficult to expand from proofs-of-concept to in vivo experiments. Until recently, the development of this technology was limited by the synthesis of oligonucleotides whose function could be significantly modulated with near-UV light. New synthetic protocols and strategies for both up- and down-regulating gene activity finally make it possible to address biological considerations. In the near future, we can expect photoresponsive DNA and RNA molecules that are relatively non-toxic, nuclease-resistant, and maintain their specificity and activity in vivo. Quantitative, laser-initiated methods for controlling DNA and RNA function will illuminate new areas in cell and developmental biology.     

RNA-aptamers that modulate the RhoGEF activity of Tiam1

Rho GTPases regulate the actin cytoskeleton and thereby control cell migration, cell morphology, cell motility, and other cellular functions. The gene product of the oncogene Tiam1 acts as a guanine nucleotide exchange factor (GEF) for the Rho GTPase Rac. Like other RhoGEFs, Tiam1 is involved in cancer progression, but it also counteracts invasion in different cancer cell types. Hence, further investigations are required to unravel the functions of Tiam1 in the context of cancer initiation and progression, which appear to be cell specific. Although RhoGEFs in general seem to be attractive therapeutic targets, not many inhibitors have been described, yet. Here we report the identification and characterization of inhibitory RNA aptamers that specifically target Tiam1. After 16 selection rounds three aptamers sharing a 15 nucleotides consensus motif were identified. The clones K91 and K11 inhibited the Tiam1-mediated activation of the GTPase Rac2 in vitro. The tightest binder K91 neither bound the Rho GEF Vav1 nor the Arf GEF Cytohesin-2. In the presence of Rac1, the binding of K91 to Tiam1 was impaired indicating that the binding motif on Tiam1 overlaps with the GTPase binding site. K91 and K11 are the first reported inhibitory molecules targeting the GEF function of Tiam1. Due to their specificity over related GEF proteins they may represent promising tools for further elucidation of the biological functions of Tiam1. We anticipated that these aptamers will prove useful in validating the ambiguous roles of Tiam1 in cancer biology.